Skin activation by acceleration of pdgf-bb activity

ABSTRACT

This method, which screens drugs that activate the skin, is characterized by causing candidate drugs to act on vascular endothelial cells, and selecting drugs that enhance the expression of PDGF-BB by the cells as a skin activation agent.

TECHNICAL FIELD

The present invention relates to skin activation by acceleration ofplatelet-derived growth factor-BB (PDGF-BB) activity.

BACKGROUND ART

Stem cells are cells that have two properties consisting of pluripotencythat allows production of cells that differentiate into a plurality ofcells, and self-replication that allows production of cells that areidentical to those cells. Stem cells derived from embryos, which are theinitial development stage of a fertilized egg, are referred to asembryonic stem cells (ES cells). Although human ES cells are expected tobe used in regenerative medicine, the production of new human ES cellsis not allowed due to ethical considerations involving the use offertilized eggs.

In recent years, attention has been focused on induced pluripotent stemcells (iPS cells) as cells demonstrating properties similar to those ofES cells. However, the production of iPS cells is associated withnumerous problems from the viewpoints of cell malignant transformation,production efficiency and the like. On the other hand, somatic stemcells, which have the ability to differentiate into specific tissue, arenot associated with ethical issues in the manner of embryonic stem cellssince they are obtained from the patient's own bone marrow or other bodytissue.

In the skin, epidermal stem cells (Non-Patent Document 1) are well-knownto be present in the epidermal basal layer, and follicular epithelialstem cells (Non-Patent Document 2) and melanocyte stem cells (Non-PatentDocument 3) have been reported to be present in regions referred to asbulge regions of hair follicles. On the other hand, although fibroblastshaving a long, narrow spindle shape are present in fibrous components ofthe skin consisting mainly of collagen, it has not yet been determinedas to whether stem cells are present in dermal fibroblasts. In addition,although skin-derived precursors (SKP) are known to exist thatdifferentiate into a plurality of cell lines such as those of fat, glia,cartilage or muscle (Non-Patent Document 4), the correlation betweendermal fibroblasts and SKP has not been determined.

Since mesenchymal stem cells, which are isolated from bone marrow asfibroblast precursors (Non-Patent Document 5), differentiate intovarious cells belonging to mesenchymal cell lines (including osteocytes,myocytes, chondrocytes, tendon cells and adipocytes), they are expectedto be applied to regenerative medicine such as the reconstruction ofbone, blood vessels and muscle. More recently, mesenchymal stem cellshave been determined to have the potential of being present in numeroustissues having mesenchymal tissue, and have been isolated from fat,umbilical cord blood and the placenta (Non-Patent Documents 6 to 8).However, the presence of mesenchymal stem cells in the dermis has yet tobe determined.

PRIOR ART DOCUMENTS Non-Patent Documents

Non-Patent Document 1: Watt, F. M., J. Dermatol. Sci., 28: 173-180, 2002

Non-Patent Document 2: Cotsarelis, G., et al., Cell, 57: 201-209, 1989

Non-Patent Document 3: Nishimura, E. K., et al., Nature, 416: 854-860,2002

Non-Patent Document 4: Wong, C. E., et al., J. Cell Biol., 175:1005-1015, 2006

Non-Patent Document 5: Pittenger, M. F., et al., Science, 284: 143-147,1999

Non-Patent Document 6: Park, K. W., et al., Cell Metab., 8: 454-457,2008

Non-Patent Document 7: Flynn, A., et al., Cytotherapy, 9: 717-726, 2007

Non-Patent Document 8: Igura, K., et al., Cytotherapy, 6: 543-553, 2004

Non-Patent Document 9: Kim, W. S., et al., J. Dermatol. Sci., 53:96-102, 2009

Non-Patent Document 10: Dalla-Favera, R., et al., Nature, 292: 31-35,1981

SUMMARY OF THE INVENTION

Mesenchymal stem cells have been determined to also be present in fat inaddition to bone marrow, umbilical cord blood and the placenta.Mesenchymal stem cells have also been determined to be present in thedermis in the same manner as subcutaneous fat located below the dermis,and further determined that they locally present at vascular sites.However, the mechanism by which these mesenchymal stem cells of thedermis and fat are locally present at vascular sites, as well as thereason why these stem cells increase or decrease with aging, areunknown. Thus, an object of the present invention is to determine thereason for increases or decreases in mesenchymal stem cells in thedermis and subcutaneous fat caused by aging, and provide a method forimproving skin condition by regulating those factors involved inmaintaining mesenchymal stem cells at these sites.

Since the number of bone marrow mesenchymal stem cells is extremely lowand sources of umbilical cord blood and placentas are limited, there arelimitations on the available sources of self-derived mesenchymal stemcells. If mesenchymal stem cells were able to be isolated from thedermis, the skin could serve as a valuable supply source of mesenchymalstem cells used in regenerative medicine and aesthetic medicine. In thisconnection, we demonstrated that mesenchymal stem cells are also presentin the dermis in the same manner as subcutaneous fat, and established amethod for isolating these mesenchymal stem cells present in the dermisand subcutaneous fat (Japanese Patent Application No. 2009-213921).Since the mechanism by which these dermal and subcutaneous fat-derivedmesenchymal stem cells are locally present at vascular sites, as well asreasons for increases and decreases in these stem cells caused by aging,are unknown, when the inventors of the present invention conductedstudies for the purpose of identifying in greater detail those siteswhere mesenchymal stem cells are present in dermis or subcutaneous fatand determining those factors that cause localization of mesenchymalstem cells, it was determined that PDGF-BB is involved. CD34-positivedermal and subcutaneous fat-derived mesenchymal stem cells decreasedwith aging, and PDGF-B gene expression also similarly decreased withaging. Ameliorative effects on wrinkles and other forms of skin aginghave been reported by injecting fat-derived mesenchymal stem cells intoaged skin (Non-Patent Document 9). Also, fat-derived mesenchymal stemcells have been determined to demonstrate an antioxidant function in theskin. On the basis thereof, skin aging can be inhibited by enhancing theexpression of endogenous PDGF-BB at vascular sites where dermal andsubcutaneous fat-derived mesenchymal stem cells are present locally, andmore specifically, in vascular endothelial cells that highly expressPDGF-BB, and maintaining a large number of mesenchymal stem cells in thedermis and subcutaneous fat.

Thus, the present application includes the following inventions:

[1] a method for screening drugs that activate skin, comprising allowinga candidate drug to act on vascular endothelial cells, and selecting thedrug that accelerates expression of PDGF-BB by the cells for use as askin activator;

[2] the method of [1], wherein the selection is carried out by measuringmRNA derived from PDGF-B in the cells by real-time polymerase chainreaction;

[3] a method for inhibiting skin aging by activating mesenchymal stemcells, and thereby activating skin, by increasing the activity or levelof PDGF-BB at a vascular site of the skin; and,

[4] the method of [3], wherein retinoic acid is applied to skin forwhich aging is desired to be inhibited;

A novel skin activator can be identified by the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 indicates expression of PDGF gene in vascular endothelial cells.

FIG. 2 indicates migration of dermal/adipose-derived stem cells inducedby PDGF.

FIG. 3 indicates localization of PDGF-BB in dermis.

FIG. 4 indicates localization of PDGF-BB in dermis.

FIG. 5 indicates the effects of PDGF-BB inhibition on accumulation ofdermal/adipose-derived stem cells in niches.

FIG. 6 indicates changes in human dermal/adipose-derived stem cellsattributable to aging.

FIG. 7 indicates changes in human dermal/adipose-derived stem cellsattributable to aging.

FIG. 8 indicates acceleration of PDGF-BB production by retinoic acid.

EMBODIMENTS OF THE INVENTION

The present invention relates to a method for screening drugs thatactivate skin, comprising allowing a candidate drug to act on vascularendothelial cells, and selecting the drug that accelerates expression ofPDGF-BB by the cells for use as a skin activator.

Platelet-derived growth factor is a growth factor involved in regulationof migration, proliferation or the like of fibroblast, smooth musclecells, glial cells and other mesenchymal cells, and is produced byvarious cells such as epithelial cells or endothelial cells. Althoughthere are at least four types of PDGF, consisting of PDGF-A, PDGF-B,PDGF-C and PDGF-D, there are also three types of isoforms (PDGF-AA,PDGF-AB and PDGF-BB) resulting from the adoption of a homo- orhetero-dimer structure as a result of the A chain and B chain formingdisulfide bonds. PDGF is known to express its physiological actionthrough PDGF receptor (PDGFR) which is a tyrosine kinase-associatedreceptor. PDGF-B gene is known and has been cloned (Non-Patent Document10).

The mesenchymal stem cells used in the present invention can be derivedfrom the dermis of all species of mammals, including humans,chimpanzees, other primates, domestic animals such as dogs, cats,rabbits, horses, sheep, goats, cows and pigs, as well as laboratoryanimals such as rats, mice and guinea pigs.

Although there are no particular limitations thereon, activation of skinas referred to in the present invention generally refers to a state inwhich metabolism of skin tissue becomes active, the turnover periodbecomes comparatively short, and tissue fatigue, atrophy and theprogression of oxidation are reduced. As a result of tissue activation,skin tightness can be maintained and the formation of wrinkles and agespots can be prevented and alleviated.

Expression of PDGF-BB gene in vascular endothelial cells may bedetermined by, for example, measuring the level of PDGF-BB. Preferably,this measurement can be carried out by a method commonly known in therelevant technical field using an antibody specific to PDGF-BB, examplesof which include various methods such as immunostaining using afluorescent substance, pigment, enzyme or the like, Western blotting orimmunoassay such as ELISA or RIA. In addition, this can also bedetermined by extracting total RNA from vascular endothelial cells, andmeasuring the amount of mRNA that encodes PDGF-B. Extraction of mRNA andmeasurement of the amount thereof are commonly known in the relevanttechnical field, and for example, quantification of RNA is carried outby quantitative polymerase chain reaction (PCR) such as real-timepolymerase chain reaction (RT-PCR). Selection of primers suitable forRT-PCR can be carried out by a method commonly known among persons withordinary skill in the art.

The inventors of the present invention found that retinoic acidaccelerates expression of PDGF-BB. Thus, selection of a skin activatorcan be carried out by investigating by means of statistical techniquesand the like whether or not a candidate drug accelerates expression ofPDGF-BB by comparing with a positive control, which uses a drug such asretinoic acid having an effect of accelerating expression of PDGF-BB,and a negative control, which uses a drug such as siRNA of PDGF-B genehaving an effect of inhibiting expression of PDGF-BB.

Moreover, the present invention also provides a method for evaluating acandidate drug for the ability to accelerate expression of apolynucleotide capable of hybridizing under highly stringent conditionswith a polynucleotide that encodes PDGF-B (SEQ ID NO: 1) in vascularendothelial cells, and selecting a candidate drug that has thataccelerating ability for use as a skin activator. Hybridization can becarried out in accordance with a commonly known method or methodapplicable thereto, such as the method described by Sambrook, et al. inMolecular Cloning 2nd Edition, Cold Spring Harbor Lab. Press, 1989, andhighly stringent hybridization conditions refer to conditions consistingof, for example, a sodium concentration of about 10 mM to 40 mM andpreferably about 20 mM, and a temperature of about 50° C. to 70° C. andpreferably about 60° C. to 65° C.

Moreover, the present invention also relates to an aesthetic method foractivating skin and thereby inhibiting skin by increasing the activityor level of PDGF-BB at vascular sites of skin to allow PDGF-BB, forwhich activity has been accelerated or expression level has beenincreased, to act on mesenchymal stem cells and activate the mesenchymalstem cells as a result thereof. As previously described, the inventorsof the present invention found that retinoic acid, and particularlytretinoic acid, accelerates expression of PDGF-BB. Thus, this method iscarried out by applying tretinoic acid to, for example, skin for whichaging is desired to be inhibited.

In addition, when a gene encoding PDGF-B in a specimen is in an inactivestate or dormant state and cells are deficient or lacking in PDGF-B as aresult thereof, the activity or level of PDGF-BB can be increased byusing a vector incorporating PDGF-BB gene in order to introduce thePDGF-B gene per se into cells. In this vector, a regulatory sequence,such as a promoter or enhancer that accelerates expression of PDGF-Bgene is preferably arranged at a location that enables it to act on thePDGF-B gene.

Either a gene insertion method using a viral vector or a non-viral geneinsertion method (Nikkei Science, April 1994, pp. 20-45; ExperimentalMedicine Special Issue, 12(15), 1994; Experimental Medicine Supplement,“Basic Technologies of Gene Therapy”, Yodosha Co., Ltd. (1996)) can beapplied for introducing the PDGF-B gene into cells. Examples of geneinsertion methods using a viral vector include methods consisting ofincorporating DNA encoding PDGF-B and inserting into a DNA or an RNAvirus such as a retrovirus, adenovirus, adeno-associated virus, herpesvirus, vaccinia virus, pox virus, polio virus or sinbis virus. Amongthese, methods using retrovirus, adenovirus, adeno-associated virus andvaccinia virus are particularly preferable. Examples of non-viral geneinsertion methods include a method consisting of intramuscularadministration of an expression plasmid (DNA vaccine method), liposomemethod, lipofectin method, microinjection method, calcium phosphatemethod and electroporation, and the DNA vaccine method and liposomemethod are particularly preferable. In addition, in vivo methods, inwhich DNA is introduced directly into cells, and ex vivo methods, inwhich a certain type of cells are extracted from a human, DNA isintroduced into the cells outside the body, and the cells are thenreturned to the body (Nikkei Science, April 1994, pp. 20-45; JapanMedicine Monthly, 36(1), 23-48 (1994), Experimental Medicine SpecialIssue, 12(15) (1994)), are used in order to allow the aforementionedgene to actually act as a pharmaceutical. In vivo methods are morepreferable. In the case of administering by an in vivo method,administration can be made, for example, intravenously, intraarterially,subcutaneously, intracutaneously or intramuscularly. In the case ofadministering by an in vivo method, administration is typically made inthe form of an injection preparation and the like, and a commonly usedvehicle may be added as necessary. In addition, in the case ofadministering in the form of liposomes or fusogenic liposomes (such asSendai virus-liposome fusion products), administration can be in theform of a liposome preparation such as a suspension, frozen preparationor centrifugally separated frozen concentrate.

EXAMPLES

The following provides a more detailed explanation of the presentinvention through examples thereof. Furthermore, the present inventionis not limited thereby.

Experimental Methods

Measurement of PDGF-B Gene Expression Level in Human

Skin Constituent Cells

The expression level of PDGF-B gene in constituent cells of human skinwas investigated by quantitative PCR. Epidermal keratinocytes KC andfollicular epithelial cells in the form of hair follicle outer rootsheath cells ORSC were cultured using EpiLife-KG2 medium (KuraboIndustries Ltd.), skin fibroblasts FB were cultured using DMEM mediumcontaining 10% FBS (Invitrogen Corp.), and human vascular endothelialcells HUVEC were cultured using EGM-2 medium (Sanko Junyaku Co., Ltd.).Each of the cells was recovered in Isogen (Nippon Gene Co., Ltd.) andtotal RNA was extracted in accordance with the protocol provided.Concentration of the purified total RNA was measured with the Nanodropnucleic acid quantification analyzer (Thermo Scientific Inc.). Eachsample was then used to synthesize cDNA in accordance with the manualprovided by Invitrogen Corp. using random primers (Invitrogen Corp.) andSuperscript II reverse transcriptase (Invitrogen Corp.). QuantitativePCR was then carried out by using the synthesized cDNA as a template andusing LightCycler FastStart DNA Master PLUS SYBR Green (RocheDiagnostics GmbH) for the reaction reagent and LightCycler (RocheDiagnostics GmbH) for the reaction device. Compositional conditions werein accordance with the Roche protocol. In addition, PCR conditionsconsisted of initial denaturation for 10 minutes at 95° C. followed bydenaturation for 10 seconds at 95° C., annealing for 10 seconds at 60°C. and extension for 10 seconds at 72° C. The sequences of the primersused are indicated below, and expression levels of PDGF gene weremeasured using software provided with LightCycler.

PDGF-A: (SEQ ID NO: 1) Forward primer: 5′-ATACCTCGCCCATGTTCTG-3′(SEQ ID NO: 2) Reverse primer: 5′-GATGCTTCTCTTCCTCCGAA-3′ PDGF-B:(SEQ ID NO: 3) Forward primer: 5′-CTTTAAGAAGGCCACGGTGA-3′ (SEQ ID NO: 4)Reverse primer:5′-CTTCAGTGCCGTCTTGTCAT-3′ PDGF-C: (SEQ ID NO: 5)Forward primer: 5′-TATATTAGGGCGCTGGTGTG-3′ (SEQ ID NO: 6)Reverse primer: 5′-ATTAAGGAGGTCCAGTGGCA-3′ PDGF-D: (SEQ ID NO: 7)Forward primer: 5′-TGGGAATCTGTCACAAGCTC-3′ (SEQ ID NO: 8)Reverse primer: 5′-CTTTTGACTTCCGGTCATGG-3′ G3PDH: (SEQ ID NO: 9)Forward primer: 5′-GCACCGTCAAGGCTGAGAAC-3′ (SEQ ID NO: 10)Reverse primer: 5'-ATGGTGGTGAAGACGCCAGT-3′

Furthermore, G3PDH was used as an internal standard, and this was usedto correct cDNA of the control group during quantification of each gene.

Evaluation of Migration Ability

Commercially available adipose-derived mesenchymal stem cells MSC werepurchased and sub-cultured in MesenPro mesenchymal stem cell medium(Invitrogen Corp.). Then, PDGF-AA, PDGF-AB or PDGF-BB (R&D Systems Inc.)was added to StemPro serum-free MSC medium (Invitrogen Corp.) in 24-wellculture plates at a concentration of 5 ng/ml to 30 ng/ml, andfibronectin-coated cell inserts (BD Bioscience Inc.) were placed thereonfollowed by seeding 50,000 MSC suspended in StemPro medium. Afterculturing overnight in a CO₂ incubator, the culture fluid was removed byaspiration. Thereafter, the cell inserts were immersed for 10 minutes inHoechist 33258-PBS solution, and the nuclei of cells adhered to the cellinsert were stained. After washing with PBS, the backs of the cellinserts were observed under a fluorescence microscope and images werecaptured. Five random images of each cell insert were captured followedby counting the number of cells that transferred to the cell insert.

Localized Sites of PDGF-BB in Human Skin Tissue Human skin tissue wasembedded in OTC Compound frozen tissue embedding medium (Sakura FinetekJapan Co., Ltd.) and 50 μm frozen sections were prepared with a Cryostatfrozen section production system (Leica Microsystems GmbH). Afterair-drying at room temperature, the frozen sections were fixed for 15minutes at room temperature using chilled acetone cooled for 15 minutesat −20° C. Then, after washing with TBS, blocking treatment was carriedout for 30 minutes with serum-free blocking reagent (Dako Corp.),followed by allowing to react overnight at 4° C. with rabbit anti-humanPDGF-BB antibody diluted 100-fold with TEST containing 3% BSA (AbcamInc.) and sheep anti-human CD31 antibody (R&D Systems Inc.). Afterwashing a total of three times consisting of twice for 40 minutes eachwith TEST and once for 40 minutes with TBS, the frozen sections wereallowed to react for 1 hour with secondary antibody labeled with Alexa488-labeled anti-sheep IgG and Alexa 594-labeled anti-rabbit IgG diluted200-fold with TBST containing 3% BSA (Invitrogen Corp.). Followingreaction, the sections were washed a total of three times consisting oftwice for 40 minutes each with TBST and once for 40 minutes with TBS,and after nuclear staining with Hoechist 33258, the sections wereobserved and imaged using an LSM5 PASCAL confocal fluorescencemicroscope (Zeiss GmbH).

Vascular Endothelial Cell Tube Formation Assay

After seeding HUVEC labeled with red fluorescent pigment (PKH26 RedFluorescent, Sigma Corp.) and MSC labeled with green fluorescent pigment(PKH67 Green Fluorescent, Sigma Corp.) in a coated 8-well chamber slideusing an in vitro angiogenesis kit, the slide was incubated for 12 hoursat 37° C. in the presence of 5% CO₂. The status of the tubes that formedwas observed using an LSM5 PASCAL confocal fluorescence microscope(Zeiss GmbH) and images were captured. In addition, mouse anti-PDGFreceptor neutralizing antibody (R&D Systems Inc.) or mouse IgGcoinciding with the isotype were used at a concentration of 5 μg/ml.

Measurement of PDGF Gene Expression Levels in Human Skin by QuantitativePCR

Human skin tissue was frozen with liquid nitrogen, and the tissue wascrushed while cooling with liquid nitrogen using a cryopress (MicrotechNichion K.K.). The samples were recovered in Isogen (Nippon Gene Co.,Ltd.) and total RNA was extracted from the skin using the protocolprovided. The expressed amount of PDGF-B gene was determined in the samemanner as previously described using the primers indicated below.

PDGF-B: (SEQ ID NO: 11) Forward primer: 5′-CCTGGCATGCAAGTGTGA-3′(SEQ ID NO: 12) Reverse primer: 5′-CCAATGGTCACCCGATTT-3′

Staining of Human Mesenchymal Stem Cells in Skin

After fixing human skin tissue for up to 1 week in formalin-phosphatebuffer solution, the tissue was embedded in paraffin using an automatedembedder (Sakura Finetek Japan Co., Ltd.). Six μm tissue sections wereprepared from the resulting human skin paraffin block with a microtome(Leica Microsystems GmbH), and the sections were affixed to an APScoated slide glass followed by flattening and drying on a flatteningtable (Sakura Finetek Japan Co., Ltd.). The prepared skin tissue slideswere de-paraffinized with xylene and subjected to hydrophilic treatmentwith a mixture of ethanol and water, and after rinsing with TBS buffer,CD34 antigen was activated by subjecting to enzymatic reaction treatmentat 37° C. for 15 minutes with 20 μg/ml Proteinase K (Roche DiagnosticsGmbH). Then, after thoroughly washing with TBST, blocking treatment wascarried out for 15 minutes with a serum-free blocking agent (DakoCorp.), followed by reacting for 1 hour at room temperature with mouseanti-human CD34 antibody (Clone QBEND-10, Abcam Inc.). After washing atotal of three times consisting of twice for 15 minutes each with TESTand once for 15 minutes with TBS, the sections were allowed to react for15 minutes with anti-mouse antibody staining reagent (Histofine MouseStaining Kit, Nichirei Corp.). Then, after washing a total of threetimes consisting of twice for 15 minutes each with TEST and once for 15minutes with TBS, the sections were reacted for 15 minutes withperoxidase-labeled streptavidin (Nichirei Corp.). Following reaction,the sections were washed a total of three times consisting of twice for15 minutes each with TBST and once for 15 minutes with TBS, followed bycarrying out a coloring reaction using Simple Stain DAB solution(Nichirei Corp.). After rinsing with distilled water withoutcounter-staining, dehydration with a mixture of ethanol and water andpenetration treatment with xylene were carried out followed by sealingusing Mount Quick (Daido Sangyo Co., Ltd.) and a cover glass. Imageswere captured with the 20× object lens of a differential interferencecontrast microscope (BX51, Olympus Corp.), and the number ofCD34-positive cells in 10 images was counted for each section.

Evaluation of PDGF-B Production Accelerating Action in

Vascular Endothelial Cells

Human vascular endothelial cells HUVEC were sub-cultured in EGM-2 medium(Sanko Junyaku Co., Ltd.), and the fourth generation cells weresuspended in Humedia-EG2 medium not containing VEGF-A (Kurabo IndustriesLtd.), followed by disseminating in a collage-coated 24-well multiplate(Asahi Glass Co., Ltd.) at a ratio of 20,000 cells and culturing for 3to 5 days at 37° C. until the cells reached confluence in the presenceof 5% CO₂. After replacing the medium with Humedia-EG2 medium (KuraboIndustries Ltd.) containing retinoic acid at 1 μm or 10 μm or DMSO usedas a solvent control, culturing was further continued for 2 days.Following completion of culturing, the culture supernatant was recoveredand PDGF-BB was quantified in accordance with the protocol providedusing Human PDGF-BB Quantikine ELISA Kit (R&D Systems Inc.). Inaddition, mRNA was extracted and purified from the cultured cells usingthe RNA extraction reagent, MagNA Pure LC mRNA HS Kit (Roche DiagnosticsGmbH), and the automated nucleic acid extraction system, MagNA Pure LC1.0 Instrument (Roche Diagnostics GmbH) in accordance with the protocolprovided. One-step quantitative RT-PCR was then carried out on thePDGF-B gene for each sample using an equal volume of mRNA as template,using the primer pair of SEQ ID NO: 9 and SEQ ID NO: 10, and usingQuantiFast SYBR Green RT-PCR Kit (Qiagen Inc.) for the reaction reagentand LightCycler (Roche Diagnostics GmbH) for the reaction apparatus.Compositional conditions were in accordance with the Qiagen protocol. Inaddition, RT-PCR conditions consisted of carrying out the RT reactionfor 20 minutes at 50° C., initial denaturation for 15 minutes at 95° C.,denaturation for 15 seconds at 94° C., annealing for 20 seconds at 60°C., and extension for 30 seconds at 72° C. Furthermore, G3PDH was usedas an internal standard, and this was used to correct the amount of mRNAof the control group.

Results

Mesenchymal stem cells have been determined to be locally present atvascular sites in human skin (Japanese Patent Application No.2009-213291). As a result of comparing expression of the four genes ofPDGF-A, PDGF-B, PDGF-C and PDGF-D in skin constituent cells for thepurpose of investigating highly expressive molecular species at vascularsites for the PDGF family known as fibroblast migration factors, PDGF-Bwas determined to be extremely high expressed in HUVEC while hardlyexpressed at all in FB (FIG. 1). In addition, PDGF-A was determined tobe expressed equally in KC, ORSC and HUVEC and at about four times thelevel of that in FB, while PDGF-C and PDGF-D were expressed atconsiderably high levels in FB (FIG. 1). Since expression of PDGF-A andPDGF-B was observed in HUVEC, it was decided to investigate the effectsof PDGF proteins PDGF-AA, PDGF-AB and PDGF-BB arising from these geneson the migration ability of mesenchymal stem cells. As a result, PDGF-BBwas determined significantly enhance the migration ability of stem cellsin comparison with PDGF-AA and PDGF-AB (FIG. 2).

Thereafter, when the localization of PDGF-BB in the dermis andsubcutaneous fat was investigated, it was found to be distributed in thesame manner as the vascular marker CD31 at sites of large vessels (FIG.3). When the distribution status at sites of large vessels was observedwith a confocal laser microscope, PDGF-BB was found to be presentoutside vascular endothelial cells and between pericytes (namely, dermalstem cells) (FIG. 4). Moreover, in contrast to nearly all mesenchymalstem cells accumulating at branched portions following their addition ina vascular endothelial cell tube formation assay, accumulation atbranched portions was inhibited in the presence of PDGF-BB receptorPDGFRβ neutralizing antibody (FIG. 5), thereby suggesting that PDGF-BBpromotes the accumulation of mesenchymal stem cells at vascular sites.When aging changes in mesenchymal stem cells and PDGF-BB wereinvestigated in human skin, it was found that the number of mesenchymalstem cells decreases with age (FIG. 6), and that expression of PDGF-Bgene similar decreases as a result of aging (FIG. 7). On the basis ofthese findings, it is thought that it possible to activate the skin byincreasing the number of mesenchymal stem cells by maintaining andaccelerating PDGF-BB.

Therefore, as a result of investigating drugs that accelerate expressionof PDGF-BB by ELISA and quantitative PCR, tretinoin (all-trans retinoicacid) was found to demonstrate concentration-dependent activity (FIG.8).

1-3. (canceled)
 4. A method for inhibiting skin aging comprising thestep of increasing the activity or level of PDGF-BB at a vascular siteof the skin, thereby causing mesenchymal stem cells to migrate toendothelial cells whose PDGF-BB activity or level has been increased, sothat the mesenchymal stem cells accumulate at the vascular site in whichsaid endothelial cells exist and activate the skin near said vascularsite.
 5. A method for screening drugs that activate skin, comprising thesteps of allowing a candidate drug to act on vascular endothelial cells,and selecting the drug that accelerates expression of PDGF-BB by thecells for use as a skin activator, wherein activation of skin isattained by migration of mesenchymal stem cells to the endothelial cellswhose PDGF-BB expression level has been increased, thereby causing themesenchymal stem cells to accumulated at a vascular site in which saidendothelial cells exist and activate the skin near said vascular site.6. The method according to claim 1, wherein the selection is carried outby measuring mRNA derived from PDGF-B in the cells by real-timepolymerase chain reaction.